The Rho-family GTPase Cdc42p regulates many aspects of cell polarity and growth in eukaryotic cells, including the organization of the actin cytoskeleton. To further examine Cdc42p function in the fission yeast Schizosaccharomyces pombe, a functional green fluorescent protein (GFP)-Cdc42p fusion protein was generated. GFP-Cdc42p was observed at the medial region of the cell at the cell-division site early in cytokinesis and remained there through cell separation, and was also localized to the periphery of the cell and to internal membranes. Unexpectedly, treatment with the actin-depolymerizing drug latrunculin-A disrupted the medial region targeting pattern, and cells deficient in the actin-binding proteins tropomyosin and profilin also did not exhibit medial GFP-Cdc42p staining. In addition, medial GFP-Cdc42p localization was eliminated in a number of cytokinesis mutants, including strains defective in assembling the medial actinomyosin ring, medial ring contraction, and septum assembly. GFP-Cdc42p targeting was less affected in mutants that formed misplaced or multiple septa. These results suggest that the localization of Cdc42p at the cell-division site was dependent upon the actin cytoskeleton and that Cdc42p may function in the interdependent processes of cytokinesis and septation.